PROJECT SUMMARY A detailed understanding of how cell fates are specified and differentiate in vertebrate tissues is fundamental to developmental and regenerative biology, and has important implications for understanding, modeling, and treating human disease. To date, we have had great success in identifying key molecular components implicated in cell fate regulation through the use of genetic screens, perturbations and fate mapping. However, a precise understanding of how cells acquire their final identities requires much deeper and unbiased examinations of the transitional cell states during differentiation. The general aim of this proposal is to combine zebrafish genetics with powerful new methods for single cell transcriptomic profiling to (Aim 1) deliver a high- resolution molecular fate map of cell differentiation during embryogenesis, (Aim 2) test hypotheses for how cell differentiation is coupled to tissue patterning in the developing spinal cord, and (Aim 3, the R00 phase) to undertake comparative molecular studies of differentiation in two distinct biological contexts: embryonic development and tissue regeneration. It is anticipated that these studies will reveal both universal and regeneration-specific mechanisms for cell differentiation, will provide significant resources to the zebrafish and larger biomedical research communities, and will help transform our approach to developmental biology, from a tradition that relied heavily on marker genes, microscopy, and qualitative observations, to an unbiased and systematic effort that interrogates the entire transcriptome at single-cell resolution. The proposed research, together with an aggressive plan for my own career development, draws on my extensive scientific background in regenerative biology and also incorporates opportunities to learn a new biological system (zebrafish) and new experimental methods, to gain exposure to a new scientific environment (the HMS Systems Biology department), and to be co-mentored by three fantastic scientists. During a K99- mentored phase, I will receive training from Drs. Sean Megason, Allon Klein, and Alex Schier, whose combined expertise in zebrafish genetics, quantitative imaging, molecular genetics, and droplet microfluidics will be invaluable as I work to build my own independent research program. As I describe below, my plan for transitioning to independence will be facilitated by annual meetings with my co-mentors to evaluate progress, and will include attendance of several workshops (including a 2- week course at Cold Spring Harbor laboratories) to develop my skills in genomics and quantitative biology. I will gain experience presenting research results in talks at international meetings and in 1-2 high-impact publications. I will also gain experience mentoring students, and will hone my lab management and grant writing skills. During my transition to independence, I will benefit from the past experiences of my co-mentors, who have all served on academic search committees and can provide practical advice as I prepare for job interviews. In addition, Dr. Alex Schier has guided 18 of his former postdocs to successful independent research positions, and his insights and feedback will be particularly valuable. My long-term career goal is to direct an independent research program aimed at understanding molecular features of cell differentiation in the contexts of embryonic development, adult tissue regeneration, and (ultimately) cell replacement therapies. So far I have achieved significant progress towards this goal in the form of research experience, successful publications, and many years of engagement with international scientific communities. I firmly believe, however, that a K99 mentored phase will help maximize my chances for success by providing access to key persons and training that would be otherwise lacking from my postdoctoral experience.